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1.
Kinetic parameters of human carbonic anhydrase B as determined from NMR linewidths of 13 C in CO 2 and HCO 3 - 总被引:1,自引:0,他引:1
The linewidths of the 13C NMR signals of CO2 and , in equilibrium aqueous solutions containing small amounts of carbonic anhydrase, are determined mainly by the rate of enzyme-induced interconversion of CO2 and . We have measured these linewidths in unbuffered solutions of human carbonic anhydrase B for several values of [CO2], at 25°C as a function of pH. From a least-squares analysis of the data, using the equations relating the linewidths to the enzyme kinetics, we have obtained values for the kinetic (Michaelis-Menten) parameters that characterize this interconversion. These preliminary results are in approximate agreement with published values for highly buffered solutions. Additionally, the results confirm that the product of the hydration reaction, and the substrate for the dehydration, is the neutral molecule H2CO3. 相似文献
2.
Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263) 总被引:3,自引:3,他引:0
We have measured the exchange of 18O between CO2 and H2O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of 18O from CO2 in the fluid outside the cells provides a method to study CO2 and HCO3− kinetics in suspensions of algae that contain carbonic anhydrase since 18O loss to H2O is catalyzed inside the cells but not in the external fluid. Low-CO2 cells of Chlorella vulgaris (grown with air) were added to a solution containing 18O enriched CO2 and HCO3− with 2 to 15 millimolar total inorganic carbon. The observed depletion of 18O from CO2 was biphasic and the resulting 18C content of CO2 was much less than the 18O content of HCO3− in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO3− into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10−4 s−1 due to our experimental errors. The Fick's law rate constant for entry of CO2 to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO2 flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO2 entry into the cell or both. The CO2 hydration activity inside the cells was 160 times the uncatalyzed CO2 hydration rate. 相似文献
3.
The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney 总被引:2,自引:0,他引:2
The steady-state kinetic parameters for the hydration of CO2 catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number kcat is 6.5 × 105 s?1 and the Michaelis constant is 7.5 mm for CO2 hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 105 s?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 108m?1s?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO2 in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO2 hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase. 相似文献
4.
Winfried Siffert Georg Fox Gerolf Gros 《Biochemical and biophysical research communications》1984,121(1):266-270
We have studied the effect of ethoxzolamide, a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10?6 M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10?11 M and 10?10M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO2 from the platelet rich plasma. These results suggest that the intracellular CO2 hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process. 相似文献
5.
Thomas H. Maren 《The Histochemical journal》1980,12(2):183-190
Summary Quantitative analysis has been made of the reactions underlying the Hansson histochemical method for carbonic anhydrase, with a view toward resolving controversies that have arisen regarding its application and specificity.The basic event is the loss of CO2 from the surface of solutions containing HCO
3
–
, PO
4
2–
and cobalt at pH 6–8. Displacement of the equilibria H2CO3 CO2 to the right elevates the pH, and at 6.8 a cobalt precipitate is formed. When tissue containing carbonic anhydrase is floated on the surface, the loss of CO2 and elevation of pH is accelerated at the enzyme site, leading to ncreased cobalt deposits. These are converted to cobalt sulphide for visualization.Study of the changes of pH and CO2 equilibria during the reaction point strongly to the fact that enzymic activity is being measured by the cobalt localization. This activity is reduced or abolished by appropriate concentrations of acetazolamide (or other sulphonamide inhibitors of carbonic anhydrase) and the powerful inorganic inhibitor, cyanate (CNO–) ion. 相似文献
6.
Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii 总被引:20,自引:17,他引:3
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We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO2, but its synthesis was induced under low concentrations of CO2 (air levels of CO2). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO2 to growth on air levels of CO2, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species. 相似文献
7.
《BBA》1986,849(3):355-365
Rapid CO2 gas exchange by Helianthus leaves was analysed kinetically using a computer model which distinguished different components of the gas exchange by different time constants. A rapid phase of CO2 uptake was ascribed to the solubilization of CO2 in all leaf compartments and to the conversion of the dissolved CO2 to HCO3− in the chloroplast stroma which contains carbonic anhydrase. From stromal ratios the stroma pH of darkened leaves was estimated to be close to 7.5. Occasionally, values as high as 8 or as low as 7 were also obtained. If fast HCO3− formation also occurs in the cytosol, pH values may be lower by about 0.3 pH units than those calculated under the assumption that carbonic anhydrase is localized in chloroplasts only. Illumination with a light intensity close to saturation of photosynthesis caused an increase in CO2 solubilization which indicated the alkalization of the chloroplast stroma by about 0.6 pH units. This is an underestimation, if the pH of cytosol decreases in the light liberating CO2 by the action of carbon anhydrase. An alkalization of the stroma by 0.6 pH units indicates the export of about 450 nmol H+/mg chlorophyll from the stroma. This forms the basis of a large transthylakoid pH gradient which drives light-dependent ATP synthesis. A pH gradient between stroma and cytosol is capable of supporting secondary gradients between these compartments in the light, such as a gradient in the ratio. On darkening, the stroma alkalization was reversed. The rate of stroma acidification was much higher in the presence of CO2 than in its absence. 相似文献
8.
Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii 总被引:32,自引:17,他引:15
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Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO2 is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO2 required for the half-maximal rate of photosynthetic O2 evolution (K0.5[CO2]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO2 fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K0.5(CO2) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO2 is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO2 from HCO3− at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO3− transport for internal accumulation might occur at the level of the chloroplast envelope. 相似文献
9.
The active species of CO2, i.e. CO2 or HCO
3
-
, formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO2 is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans. 相似文献
10.
Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C(i) Cells of Chlamydomonas: Studies Using O Exchange with C/O Labeled Bicarbonate
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By measuring 18O exchange from doubly labeled CO2 (13C18O18O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [Ci]) or air enriched with 5% CO2 (high Ci). Intact low Ci protoplasts had a 10-fold higher carbonic anhydrase activity than did high Ci protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of 18O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low Ci cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low Ci than in high Ci preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment. 相似文献
11.
Samer Al-Samir Maximilian Prill Claudiu T. Supuran Gerolf Gros Volker Endeward 《Journal of enzyme inhibition and medicinal chemistry》2021,36(1):1602
We have studied the CO2 permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs 18 O-labelled CO2. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO3− permeability. For normal rat erythrocytes, we find at 37 °C a CO2 permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10−3 cm/s. We studied whether the rat erythrocyte membrane possesses protein CO2 channels similar to the human red cell membrane by applying the potential CO2 channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO2 permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO2 channels that are responsible for at least 50% of its CO2 permeability. 相似文献
12.
Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis 总被引:10,自引:6,他引:4
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In C4 plants carbonic anhydrase catalyzes the critical first step of C4 photosynthesis, the hydration of CO2 to bicarbonate. The maximum activity of this enzyme in C4 leaf extracts, measured by H+ production with saturating CO2 and extrapolated to 25°C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C3 leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO2 concentrations was apparently just sufficient to prevent the rate of conversion of CO2 to HCO3− from limiting C4 photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities. 相似文献
13.
The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co2+, Cu2+, and Fe3+, increased the enzyme activity, whereas CA activity was inhibited by Pb2+, Hg2+, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO2 to CaCO3. The maximum CO2 sequestration potential was achieved with immobilized CA (480 mg CaCO3/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO2 sequestration. 相似文献
14.
J. A. Raven 《Plant, cell & environment》2001,24(2):261-265
A model is presented which quantifies a possible role for the carbonic anhydrase in the mitochondrial matrix of Chlamydomonas reinhardtii which incorporates the observation that the expression of this enzyme is increased under growth conditions in which the expression of the carbon dioxide-concentrating mechanism is increased. It is assumed that the inorganic carbon enters the cytosol from the medium, and leaves the cytosol to the plastids, as HCO3− and that there is negligible carbonic anhydrase activity in the cytosol. The role of the mitochondrial carbonic anhydrase is suggested to be the conversion to HCO3– of the CO2 produced in the mitochondria in the light from tricarboxylic acid cycle activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity which is not suppressed by the carbon concentrating mechanism. If there is a HCO3− channel in the inner mitochondrial membrane then almost all of the inorganic carbon leaves the mitochondria as HCO3−, thus limiting the potential for CO2 leakage through the plasmalemma. This mechanism could increase inorganic C supply to ribulose bisphosphate carboxylase-oxygenase by some 10% at the energetic expense of less than 1% of the total ATP generation by plastids plus mitochondria. 相似文献
15.
Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas 总被引:5,自引:5,他引:0
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Inorganic carbon (Ci) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO2. Both air-grown cells, that have a CO2 concentrating system, and 5% CO2-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (Ci) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO2-grown cells also accumulated some Ci, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO2 fixation by high CO2-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO2-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase. 相似文献
16.
Expression of a carbonic anhydrase gene is induced by environmental stresses in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) 总被引:2,自引:0,他引:2
Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO3 and Na2CO3), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses.
Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO3, 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in
rice. 相似文献
17.
Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380) 总被引:10,自引:7,他引:3
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We report the changes in the concentrations and 18O contents of extracellular CO2 and HCO3− in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring 18O exchange between CO2 and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO2 hydration rate in different samples of cells that were grown on bubbled air (low-CO2 conditions). This activity was only weakly inhibited by ethoxzolamide with an I50 near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO2-starved cells there is considerable generation of CO2 from intracellular stores, a factor that can cause errors in measurement of net CO2 uptake unless accounted for. It was demonstrated that use of 13C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO2 readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO3− was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO3− from Synechococcus. 相似文献
18.
Mass spectrometric measurements of dissolved free 13CO2 were used to monitor CO2 uptake by air grown (low CO2) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the 13CO2 concentration in the medium to close to zero. Similar results were obtained with low CO2 cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO2 uptake revealed that the removal of CO2 from the medium in the light was due to selective and active CO2 transport rather than uptake of total dissolved inorganic carbon. In the light, low CO2 cells and protoplasts incubated with carbonic anhydrase took up CO2 at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO2 uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO2 cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO2 compensation point. During the linear uptake of CO2, low CO2 cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O2 evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O2 evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO2 uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO2 had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O2 evolution. These results show low CO2 cells of Chlamydomonas are able to transport both CO2 and HCO3− but CO2 is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO2 from the dehydration of HCO3−. 相似文献
19.
Inorganic carbon uptake was investigated in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Mass spectrometric and potentiometric assays indicated that both species lacked external carbonic anhydrase (CA). The presence of internal CA was demonstrated by potentiometric assay and by the inhibition of photosynthesis upon the addition of 500 μM ethoxyzolamide a membrane‐permeable inhibitor of CA. The capacity for bicarbonate transport was investigated by comparing the calculated rate of spontaneous CO2 formation at pH 8.2 and 25°C with the rate of photosynthesis after the addition of 100 μM NaHCO3. Both species appeared to have a very limited capacity for direct bicarbonate uptake. Monitoring of CO2 and O2 fluxes in both species by mass spectrometry demonstrated a rapid uptake of CO2 on illumination, to concentrations below the CO2 equilibrium concentration, indicating an effective selective uptake of CO2. This dependence of photosynthesis on free CO2 alone suggests that these species are CO2 limited in their natural environment because the CO2 concentration of seawater is very low. 相似文献
20.
Crab gill carbonic anhydrase is shown to facilitate the excretionof carbondioxide across isolated perfused gills. A techniquefor perfusing crab gills and assessing the metabolic viabilityof perfused gills is also described in detail. The techniqueis used to follow the disappearance of 14C label as HCO3and CO2 from internal perfusate passing through the gill. Theexcretion of the label increases with the flow rate of the externalperfusate across the outside of the gills. The addition of carbonican hydrase to the internal perfusate results in a two- to fourfoldincrease in the excretion of label while Diamox (acetazolamide)treatment decreases the excretion of label by half. It is alsosuggested that carbonic anhydrase, present in muscle tissuesof crabs, minimizes the disequilibrium of the hemolymph CO2system as metabolically produced CO2 leaves the tissues andenters the hemolymph. Parallels are drawn between the presenceof carbonic anhydrase in the crab gill system and the presenceof this enzyme in the respiratory organs of both aquatic andterrestrial animals. 相似文献